Vanilla is extracted from the cured pods of the flowers of the vanilla vine, a member of the orchid family. During the curing process, a large number of flavor compounds are formed which impart to the extract the pleasing aroma and well balanced organoleptic properties characteristic of vanilla. While vanilla is highly prized for use in flavoring a broad array of foodstuffs, its use is restricted by high cost stemming from the complex, low yielding methods associated with its manufacture. Vanillin (3-methoxy-4-hydroxybenzaldehyde) is one of the principle components responsible for the characteristic aroma and flavor of vanilla extract. Synthetic vanillin, most often produced by the treatment of sulfite waste liquors from paper mills, is typically used as a low cost substitute for vanilla and indeed may even be present as an adulterant in vanilla extract. Because of its origin and method of manufacture, vanillin derived from sulfite waste liquor is not considered to be a natural food component nor may it be so labeled in the U.S. A process for the production of natural vanillin via the bioconversion of natural vanillin precursors by a low cost process would therefore have great value and utility.
It is well known that vanillin is formed in small quantities from aromatic compounds known to be precursors in the biosynthesis of lignin (see Rahouti, Mohammed, et al., Appln. Environ. Microbiol., 55:2391-8, Sep., 1989; Tadasa, K., Agric. Biol. Chem. 41:925-9, 1977; Sutherland, J. B. et al., Can. J. Microb., 29:1253-7, 1983). Trans-ferulic is a common precursor in the biosynthesis of lignin as well as a lignin degradation product. The metabolism of ferulic acid by soil microorganisms typically leads to the production of vanillin but the vanillin so formed is further converted to vanillic acid and/or vanillyl alcohol which in turn are typically transformed to other degradation products such as protocatechuic acid, guaiacol, hydroquinones, catechol, ring-cleavage products, etc. Thus, it is generally recognized that vanillin is an intermediate in the overall scheme for the biodegradation of ferulic acid and ferulic acid precursors so that only small amounts of vanillin accumulate when ferulic acid is biodegraded by soil microorganisms. There are a large number of references which report on the metabolism of aromatic compounds by soil microorganisms (e.g., Pullularia pullulans Fusarium sp., Aspergillus niger, Bacillus sp., Pseudomonas sp., Nocardia sp., see Borneman, W. S. et al., Appl. Microbiol. Biotechnol. 33:354-51, 1990; Eggeling, L. et al., Arch. Microbiol. 126:141-8, 1980) but none stress the potential for using such microorganisms for vanillin synthesis because vanillin accumulates only small quantities as an intermediate product.
U.S. Pat. No. 5,057,424 to Knuth and Sahai teaches callus cells which are independent of and unattached to differentiated plant tissue which are derived from plant tissue. The callus cells are capable of growth in cell culture without forming differentiated cell structures. The vanilla plant callus cells secrete vanillin and related vanillin flavor components into the culture medium in which they are cultured. The patent specification identifies three principal variables in the liquid culture medium which can be adjusted to improve the yield of vanillin and related components. These variables are (a) nutrient, (b) plant hormones, and (c) vanilla component precursors. The inventors report that experiments leading to the Knuth et al. invention suggest that phenylalanine and ferulic acid, two metabolic precursors of vanillin, produced little enhancement in vanillin production by the callus cell culture. The copending application to Knuth and Sahai which issued as U.S. Pat. No. 5,068,184 discloses a process to produce a vanilla flavor composition by suspending vanilla-plant callus cells in tissue culture under conditions that promote the secretion of vanilla flavor components by the cells into the culture medium. Both Knuth and Sahai patents identified above are herein incorporated by reference into the present application.